UNIVERSIDADE FEDERAL DE PELOTAS Programa de Pós-Graduação em Ciência e Tecnologia Agroindustrial

UNIVERSIDADE FEDERAL DE PELOTAS

Programa de Pós-Graduação em Ciência e Tecnologia

Agroindustrial

Tese

COMPARAÇÕES FILOGENÔMICAS ENTRE CEPAS DE Listeria

monocytogenes ISOLADAS DE DIFERENTES FONTES E REGIÕES

GEOGRÁFICAS

Élen Silveira Nalério

Pelotas, 2009

ÉLEN SILVEIRA NALÉRIO

COMPARAÇÕES FILOGENÔMICAS ENTRE CEPAS

DE Listeria monocytogenes ISOLADAS DE DIFERENTES FONTES E REGIÕES GEOGRÁFICAS

Tese apresentada ao Programa de Pós-Graduação em Ciência e Tecnologia Agroindustrial da Universidade Federal de Pelotas, como requisito parcial à obtenção do título de Doutor em Ciências (Microbiologia de Alimentos).

Orientador: Prof. Dr. Wladimir Padilha da Silva Co-Orientador (es): Dr. Brendan William Wren Dr. Richard Stabler

Banca examinadora:

Prof. Dr. Wladimir Padilha da Silva Prof. Dr. Fábio Pereira Leivas Leite Profa Dra Márcia Monks Jantzen Profa Dra Amanda de Souza da Motta

À minha família Mãe, Pai, Natália e Leonardo

AGRADECIMENTOS

A Deus, por estar sempre junto a mim, me guiando.

A minha família, mãe, pai, Natália e Léo, por serem a base de tudo e, incansavelmente, estarem ao meu lado, incentivando e torcendo pelos meus ideais.

A grande família Nalério, onde todos, mesmo que indiretamente, sempre estiveram presentes na minha vida me apoiando, principalmente, minha “irmã” Ivna, que desde a infância sonhou comigo o futuro.

As minhas queridas amigas Márcia e Karla que sempre me apoiaram e fizeram presentes de alguma forma.

Ao Paulo Henrique, que apesar de ter entrado na minha vida recentemente, tem me acolhido de forma muito especial e, com seu carinho, tem demonstrado o quanto acredita em mim.

Ao meu orientador e amigo, Wladimir Padilha da Silva, que desde a graduação me mostrou uma conduta profissional exemplar e, acima disso, me proporcionou crescer.

Aos amigos do Laboratório de Microbiologia de Alimentos Prof. Celso, Marcelo, Carol, Rodrigo, Andréia, Milena, Ana Eucares, Márcia Mata, Kátia, Denise, Júlia, Greici, Valmor, Simone e Janaina.

Aos professores e funcionários do Departamento de Ciência e Tecnologia Agroindustrial.

Aos amigos da London School of Hygiene and Tropical Medicine, especialmente Ozan Gudogdu, Suaad Al-Jaberi, Pornpan Pumirat e Pippa Strong pela grande amizade.

Ao Brendan Wren e Richard Stabler pela disponibilização de estrutura, material e de conhecimentos científicos, além disso pela confiança depositada em mim. À Universidade Federal de Pelotas e ao Programa de Pós-Graduação em Ciência e Tecnologia por proporcionarem a realização do curso.

À CAPES (Coordenação de Apoio a Pesquisa em Nível Superior) pela concessão da bolsa de estudos no Brasil e no exterior.

RESUMO

NALÉRIO, ÉLEN SILVEIRA

.

Pós-Graduação em Ciência e Tecnologia Agroindustrial. Universidade Federal de Pelotas, Pelotas.

Listeria monocytogenes é o agente causador da listeriose, uma infecção severa que pode cursar com sintomas que variam desde gastroenterites, meningites e até mesmo a morte. De fato, o desenvolvimento da doença pode ser relacionado a determinados sorotipos/linhagens das cepas de Listeria. Análises moleculares dos diferentes sorotipos/linhagens de L. monocytogenes, demonstraram que esta espécie é amplamente diversa, a qual pode ser agrupada em três linhagens. O estudo completo de genomas, baseado na técnica de microarray, pode ser empregado para estudar a relação filogenética entre cepas de Listeria tanto em nível de espécie, quanto em nível de sorotipo. Não obstante, a técnica de microarray visa evidenciar as diferenças no potencial patogênico e/ou adaptativo das cepas. O objetivo deste estudo foi a comparação filogenética entre cepas de L. monocytogenes isoladas de diferentes fontes. Foram analisadas 99 cepas de L. monocytogenes de diferentes origens geográficas (Brasil, Dinamarca, Áustria, Irlanda, Estados Unidos da América e fontes desconhecidas), incluindo cepas clínicas (humanas e animais), de alimentos e de indústrias alimentícias. O DNA das cepas teste foi hibridizado em DNA microarrays de L. monocytogenes baseado em seqüências do genoma de L. monocytogenes EGD-e. Os protocolos para marcação e hibridização do DNA seguiram as recomendações de Dorrell et al., (2001). A aquisição de dados, o processamento e as comparações filogenômicas foram realizadas conforme previamente descrito por Stabler et al. (2006). Comparações filogenômicas agruparam as cepas de L. monocytogenes em dois clades centrais, os quais são representativos das duas principais linhagens desta espécie. Além disso, cada um destes clades foram sub-divididos em mais dois sub-clades. A formação dos clades foi independente da origem geográfica das cepas, com exceção do clade contendo cepas persistentes (cepas que persistem no ambiente de processamento de alimentos), onde nenhuma cepa Brasileira esteve presente. Foram identificados 18 genes específicos para as cepas da linhagem I (sorotipos 1/2a e 1/2c). Esses genes são relacionados ao metabolismo de carboidratos, sistema regulatório two component, complexo de transporte ABC e aos genes bvrB e bvrC. A grande maioria das cepas persistentes se agrupou no mesmo clade pertencente à linhagem I. Foi obtido um conjunto de genes únicos pertencentes exclusivamente às cepas persistentes de L. monocytogenes, os quais sugerem serem os responsáveis pelo perfil adaptativo destas cepas. Os genes são envolvidos em resistência ao estresse e são relacionados ao transporte e metabolismo de carboidratos, processamento de informação ambiental, mecanismos de transdução de sinais, proteína de superfície celular, transporte e metabolismo de aminoácidos, transporte e metabolismo de nucleotídeos, tradução, biogênese de parede celular, replicação, recombinação e reparo, transporte de pequenas moléculas similar ao transportador ABC, metabolismo de lipídios e de função desconhecida. Dos 14 genes de virulência listados a

maioria deles esteve presente em todas as cepas de L. monocytogenes estudadas, com exceção dos genes inlE e inlG. Estes dados sugerem que, apesar das distintas origens de isolamento, a variabilidade genética das cepas de L. monocytogenes é direcionada para adaptação ambiental, ao invés da diferenciação visando virulência.

Palavras-chave: Listeria monocytogenes, comparações filogenômicas, genes de virulência, genes de persistência, microarray.

ABSTRACT

NALÉRIO, ÉLEN SILVEIRA

.

Listeria monocytogenes STRAINS ISOLATED FROM DIFFERENT SOURCES AND GEOGRAPHIC REGIONS. 2009. 65f. Tese (Doutorado).

Programa de Pós-Graduação em Ciência e Tecnologia Agroindustrial. Universidade Federal de Pelotas, Pelotas.

Listeria monocytogenes is the causative agent of listeriosis which may cause a range of diseases from gastroenteritis, meningitis and death. In fact, disease outcome can be related to strain serotype/lineage thus molecular analyses has demonstrated that L. monocytogenes is a highly diverse species which can be grouped into three lineages. Whole-genome microarray can be employed to study phylogenetic relationships among Listeria strains either species or serotype level, in addition to demonstrate differences on their virulence potential and/or environmental adaptation. The aim of this study was the whole genome comparison of L. monocytogenes strains from different origins. Ninety-nine L. monocytogenes strains from different geographical origins (Brazil, Denmark, Austria, Ireland, USA and unknown), including clinical strains (humans and animals), food and food industries strains were analysed. DNA from all strains were competitively hybridized on to a L. monocytogenes DNA microarray based on the whole-genome sequence L. monocytogenes EGD-e. DNA labeling and hybridization protocol were followed according to Dorrell et al., (2001). Data acquisition, processing and comparative phylogenomics were performed as previously described by Stabler et al. (2006). Comparative phylogenomics clustered the L. monocytogenes strains into two central clades which is representative of the two main lineages of this species. In addition each of these clades were divided into two further subclades. Clade formation was independent of the geographical origin of strains with the exception of the clade containing persistent strains (strains that persist in food-processing environment), where none of the Brazilian strains were present. It was found 18 specific genes for lineage I strains (1/2a and 1/2c serotypes). These genes are related to carbohydrate metabolism, two component regulatory system, ABC transporter complex and bvrB and bvrC genes. Significantly all persistent strains clustered together in the same lineage I clade. We achieved a set of unique genes belonging exclusively to L. monocytogenes persistent strains pointing to be responsible for its adaptation profile. The genes are involved in stress resistance and are related to carbohydrate transport and metabolism, environmental information processing, signal transduction mechanisms, cell surface protein, amino acid transport and metabolism, nucleotide transport and metabolism, translation, cell wall biogenesis, replication, recombination and repair, transport of small molecules similar to ABC transporter, metabolism of lipids and unknown function. Interestingly from 14 virulence listed genes most of them were present in all studied L. monocytogenes strains with exception of inlE and inlG genes. These findings indicate that genetic variability of L. monocytogenes strains point to niche adaptation instead virulence differentiation despite of different origins. Persistent strains clustered suggesting genetic origin to survival in this environment.

Keywords: Listeria monocytogenes, phylogenomic comparisons, virulence genes, persistent genes.

Sumário

1 INTRODUÇÃO... 12

2 TÍTULO 1: Whole genome comparison of Listeria monocytogenes isolated from several countries and sources………... 15 1. ABSTRACT……… 16

2. INTRODUCTION………..………... 17

3. MATERIALS AND METHODS………... 18

3.1 Strains collection………..………. 18

3.2 Genomic DNA extraction... 18

3.3 Microarray design……….. 18

3.4 DNA labelling and hybridization... 20

3.5Data acquisition, processing and comparative phylogenomics……...……….. 20

4. RESULTS AND DISCUSSION... 21

4.1 Overall microarray analysis………. 21

4.2 Comparative phylogenomics of core genes……… 24

4.3 Comparative diversity among geographical origins, sources and serotypes of L. monocytogenes strains………... 25

4.4 Virulence genes………... 27

5. REFERENCES……… 28

3 TÍTULO 2: Comparative phylogenomics of Listeria monocytogenes reveals specific genes related to adaptation profile…..………. 39

1. ABSTRACT……… 40

2. INTRODUCTION……….. 41

3.1 Strains collection………..………. 42

3.2 Genomic DNA extraction... 42

3.3 Microarray design……….. 44

3.4 DNA labelling and hybridization... 44

3.5 Data acquisition, processing and comparative phylogenomics…….. 44

4. RESULTS AND DISCUSSION... 45

4.1 Persistent or adaptive genes identification………….……...……… 47

5. REFERENCES……… 52

4 CONCLUSÕES………. 63

1 INTRODUÇÃO

As bactérias pertencentes ao gênero Listeria possuem a característica de serem difundidas na natureza, uma vez que estão presentes no solo, água, alimentos e na microbiota intestinal do homem e dos animais (SEELIGER, 1961; FARBER AND PETERKIN, 1991; ROCOURT & COSSART, 1997). Listeria monocytogenes é um microrganismo de grande interesse em saúde pública e também para as indústrias alimentícias (GUDMUNDSDÓTTIR et al., 2005), embora tenha sido reconhecido como um patógeno humano antes dos anos 70 (GOMBAS et al., 2003), somente nas três últimas décadas foi associado aos alimentos e classificado como patógeno de origem alimentar. É o agente causador da listeriose, uma infecção severa, a qual apresenta taxa de mortalidade entre 20-30% (DUSSURGET, PIZZARO-CERDA & COSSART, 2004; WALLS & BUCHANAM, 2005).

Alimentos contaminados são as maiores fontes de infecção de Listeria, e o trato gastrointestinal é o sitio primário de entrada da bactéria no organismo do hospedeiro (VASQUES-BOLAND et al., 2001). Qualquer cepa de L. monocytogenes pode ser considerada potencialmente patogênica para os humanos. Entretanto, várias observações sugerem que L. monocytogenes apresenta virulência heterogênea, em função de seus sorotipos, os quais estariam associados, de alguma forma, com o potencial de patogenicidade do microrganismo (JACQUET et al., 2002). Dos 13 sorotipos conhecidos, somente três (1/2a, 1/2b e 4b) têm sido relacionados a 90% dos surtos e casos esporádicos de listeriose (CABRITA et al., 2004), sendo as cepas do sorotipo 4b mais comumente isoladas em surtos, e cepas dos sorotipos 1/2a e 1/2b em casos esporádicos (FARBER & PERTERKIN, 1991; LIANOU et al., 2006).

Os avanços nas tecnologias de biologia molecular permitiram o desenvolvimento de técnicas eficazes para a análise em apenas, um único experimento, de um grande número de genes bacterianos (DORRELL, HINCHLIFFE & WREN, 2005). Assim, a técnica de microarray fornece uma revolução para estudos de diversidade genética de patógenos como L. monocytogenes (CALL, BORUCKI & LOGE, 2003; RUDI, KATLA & NATERSTAD, 2003; GARAIZAR, REMENTERIA & PORWOLLIK, 2006).

Os microarray são pequenos chips de DNA aderidos em lâminas onde milhares de seqüências de DNA ou cDNA são arranjados, representando genes conhecidos ou seqüências inteiras de um gene com todas suas possibilidades de mutação (BENEDETTI et al., 2000; DORRELL, HINCHLIFFE & WREN, 2005). Desta forma, em uma única lâmina é possível a deposição de milhares de seqüências, que são imobilizadas de forma organizada e em posições conhecidas (DORRELL, HINCHLIFFE & WREN, 2005; GARAIZAR, REMENTERIA & PORWOLLIK, 2006).

A aplicação de DNA microarray em subtipagem epidemiológica microbiana é ainda esporádica, este fato se deve, em parte, a pouca disponibilidade de genomas completos para a execução dos arranjos e também pelo elevado custo do método (GARAIZAR, REMENTERIA & PORWOLLIK, 2006). Estes micro-arranjos são baseados no genoma de uma cepa bacteriana completamente seqüenciada e em uma coleção de sondas de DNA fixadas em uma superfície sólida. Assim, em um único experimento de hibridização, o status de ausência\presença de todos os genes de um patógeno seqüenciado e de uma requerida cepa pode ser examinada (GARAIZAR, REMENTERIA & PORWOLLIK, 2006).

A disponibilidade do genoma completo de L. monocytogenes (GLASER et al., 2001) permitiu o desenvolvimento de DNA microarray. Com isso, muitas estratégias baseadas nesta técnica podem ser desenvolvidas para diferenciar L. monocytogenes entre seus sorotipos e linhagens filogenéticas (NITHINGALE et al., 2005; HAIN, STEINWEG & CHAKRABORTY, 2006). Além disso, seguindo a expansão de dados oriundos das análises de biologia molecular, uma série de aplicações práticas podem ser estabelecidas para esta técnica de DNA microarray (PANDA et al., 2003), principalmente, aquelas propondo contribuir para a elucidação dos mecanismos que determinam as diferenças

entre cepas epidemiologicamente relacionadas e também para compreender a adaptação, persistência e/ou virulência de L. monocytogenes (DOUMITH et al., 2006).

TÍTULO 1: Whole genome comparison of Listeria

Whole genome comparison of Listeria monocytogenes isolated

from several countries and sources

Nalério, E. N.*; Silva, W. P.*; Stabler**, R. A.; Wren, B. W.**

*Department of Agro-industrial Science and Technology, Laboratory of Food Microbiology, University Federal of Pelotas, Pelotas, Brazil

**Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom

1. ABSTRACT

L. monocytogenes is the causative agent of listeriosis which may cause a range of diseases from gastroenteritis to death. The disease outcome can be related to strain serotype/lineage thus molecular analyses has demonstrated that L. monocytogenes is a highly diverse species which can be grouped into three lineages. Whole-genome microarray can be employed to study phylogenetic relationships among Listeria strains either species or serotype level, in addition to demonstrate differences on their virulence potential and/or environmental adaptation. The aim of this study was to compare the whole genome of L. monocytogenes strains from different origins. Thus 99 L. monocytogenes strains from different geographical origins (Brazil, Denmark, Austria, Ireland, USA and unknown), including clinical strains (humans and animals), food and food industries strains were analysed, plus one L. innocua strain. The Bayesian algorithm allowed the clustering of L. monocytogenes strains into two central clades which is representative of the two main lineages of this species. The clustering were independent of the geographical origin of strains, the exception was the clade gathering the vast majority of probable persistent strains, where none of the Brazilian strains were present. It was found 18 specific genes for lineage I strains (1/2a and 1/2c serotypes). These genes are related to carbohydrate metabolism, two component regulatory system, ABC transporter complex and bvrB and bvrC genes. Interestingly from the 14 virulence listed genes most of them were present in every studied L. monocytogenes strain with exception of inlE and inlG genes. These results demonstrate that virulence genes involved in L. monocytogenes pathogenicity are conserved among the different strains.

2. INTRODUCTION

Listeria monocytogenes is the causative agent of listeriosis, which may cause a range of diseases from gastroenteritis to death (Schlech, 1998). Although listeriosis has low incidence in comparison with other food-borne diseases, it is a great public health concern due its mortality rate which is around 20-30% (Mead et al., 1999). Importantly, the epidemiology of listeriosis in Europe is changing and the incidence is increasing (Goulet et al., 2008). In the last five years the incidence has increased 59% in the European countries (EFSA, 2007). These increases have been reported in Belgium, Denmark, Germany, Finland, France and Switzerland (Goulet et al., 2008). In England and Wales morbidity rate has increased up to 44% in the patients for whom outcome data was available. The reason for this upsurge is still uncertain but it might be due in part to improvement in the surveillance network systems (Gillespie et al., 2006).

Interestingly, listeriosis has not been frequently reported in developing countries despite the fact that the bacterium has been widely isolated in food and food-processing plants (Lima et al., 2005; Nalério et al., 2009; Hellstorm et al., 2010; Myia et al., 2010). First impressions may suggest a divergence on virulence according to geographic regions; however this situation is probably due to inappropriate health surveillance networks. In Brazil, for instance, like many developing countries, notification of listeriosis is not compulsory therefore few has been reported as a consequence few official statistic data, which makes comparison to epidemiologic data from European countries, United States of America and Canada difficult. However in a study carried out in Brazil with 3,112 Listeria strains isolated from 1971 to 1997 reported that 74.9% of those strains were isolated from food, 9.5% from environment, 7.9% from human cases and 7.8% from animal cases. L. monocytogenes was isolated in 24.9% of all samples and in 96% of strains isolated from humans moreover most of those strains were related to pathological processes (Hofer et al., 2000).

Although nearly all L. monocytogenes strains have been considered pathogenic, it appears that are not all strains capable of developing disease. In

fact, disease outcome can be related to strain serotype/lineage. There are 13 recognised L. monocytogenes serotypes but only three (4b, 1/2a and1/2b) have been associated with approximately 95% of cases and outbreaks of listeriosis (Schuchat et al., 1991; Kathariou, 2002). Molecular analyses as like AFLP (Amplified Fragment Length Polymorphism) (Aarts et al., 1999), MEE (Multilocus Enzyme Electrophoresis) (Bibb et al., 1989) and PFGE (Pulsed-field Gel electrophoresis) (Brosh et al., 1994) demonstrated that L. monocytogenes is a highly diverse species and which can be grouped into three lineages. Lineage I (consisting of serotypes 1/2a, 3a, 1/2c and 3c), lineage II (1/2b, 3b, 4b, 4d, 4e and 7) and lineage III (4a and 4c) (Doumith et al., 2004a; Nightingale et al., 2005). This classification was additionally subdivided in 6 phylogenetic groups, lineage I.1 (serotypes 1/2a and 3a), I.2 (1/2c and 3c), II.1 (4b, 4d and 4e), II.2 (1/2b, 3b and 7), III.1 (4a) and III.2 (4c) (Doumith et al, 2004b). Furthermore it has been suggested that lineage I represent an environmental adapted lineage whilst lineage II strains represent a human host adapted lineage (Wiedmann, 2002).

Powerful and highly discriminatory methods are extremely necessary for understanding the divergences on epidemiology and evolutionary relationships about L. monocytogenes. Consequently microarray is efficient on comparing whole-genomes, allowing a deep insight into genetic content of large strain collections (Dorrell et al., 2005).Thus the construction of a whole-genome microarray (Hinds et al., 2002) to study this associations among Listeria strains either species or serotype level may fulfill this lacking of knowledge. Comparative phylogenomics employing Bayesian mathematical analyses is able to determine phylogenetics relationships, permitting robust approach into genetic relatedness of bacterial pathogens (Dorrel et al., 2005; Champion et al., 2005). Additionally this technique may be used as an alternative to characterise pathogenic isolates (Call et al., 2003a) by subtypification and also providing an understanding in the molecular phylogeny and evolution of L. monocytogenes strains giving information about transmission, pathogenesis and niche adaptation (Call et al., 2003b; Nightingale et al., 2005).

Strains collection

The 100 strains used in this study were from diverse countries and sources, to date: 47 strains from Brazil, 10 from Denmark, 08 from Austria, 6 from Ireland, 1 from Unites States and 26 from unknown origins (Table 1). These strains were isolated from food (58), equipment and environment of industries (16), clinical cases both humans and animals (14), asymptomatic animal carriers (2), environment (6) and unidentified sources of isolation (5). On this collection is also included one L. innocua strain.

Genomic DNA (gDNA) extraction

The genomic DNA extraction was carried out with Wizard (Promega) purification kit and its quality and quantification was performed by 1% agarose gel electrophoresis and GeneQuant spectrophotometer (Amersham Pharmacia), respectively. The gDNA samples were kept at 4°C to minimize damage which can be caused by freeze thawing.

Microarray Design

From 2944 annotated sequences of the whole-sequenced strain EGD-e (Glaser et al., 2001), 2855 were included in the design process. The L. monocytogenes arrays were developed and printed by BµG@S (Bacterial Microarray Group at St. Georges’s Hospital Medical School), Department of Cellular and Molecular Medicine, St George’s Hospital Medical School, London (Hinds et al., 2002). Briefly, ten pairs of gene-specific primers were designed to each sequence by applying PRIMER3 (Rozen & Skaletsky, 2000). Primers were designed to have a length of 20-25 base pairs (bp), melting temperature between 50°C and 80°C and amplicon ranging in size from 100 to 800 bp in length with an optimum size of 600 bp. The PCR primers were selected based on the BLAST similarity of the predicted PCR product to other genes on the microarray. Products with no similarity or least similarity were selected to ensure the least possible cross-hybridization on the microarray. The PCR products are spotted at a high density on poly-L-lysine-coated glass microscope

slide, using a MicroGridII (BioRobotics) arraying robot. Array design is available from ArrayExpress database (www.ebi.ac.uk/arrayexpress accession number A-BUGS-19).

DNA labelling and hybridization

For each microarray slide, 5µg of both test genomic DNA labelled with cyanine dye Cy3 Fluorolink™ (Amersham Bioscience) and L. monocytogenes EGD-e DNA labelled with cyanine dye Cy5 Fluorolink™ (Amersham Bioscience) was used in the microarray hybridization. The L. monocytogenes EGD-e is a standard strain (control) used as general reference for all hybridizations as previously described by Dorrell et al. (2001). Microarray slides were prehybridized into a Coplin jar within a solution which consist in 3.5 X SSC (1X SSC - 0.15M NaCl plus 0.015M Sodium Citrate), 0.1% SDS (Sodium Dodecyl Sulfate) and 10mg/mL BSA (Bovine Serum Albumin) at 65ºC for 20 minutes. After this the slides were rinsed thoroughly in distillate water followed for rinsing in isopropanol 1 minute each, centrifuged and kept way from light until the moment of hybridization.

After DNA labelling, test and control DNA, Cy3-labelled DNA and Cy5-labelled DNA, respectively, were mixed together and then purified with MinElute Purification Kit (Qiagen). Just before the hybridization the solution was mixed with 20X SSC and 2% SDS followed for denaturation at 95ºC for 2 minutes, applied on microarray slide and covered with 22 x 22mm LifterSlips (Eyrie Scientific), hybridized overnight sealed in a humidified hybridization chamber (Telechem International) and immersed in a bath at 65ºC for 16-20 hours. Afterwards, the slides were washed first in a pre-heated at 65ºC Wash A solution (1x SSC, 0.05%SDS and distillated water) for 2 minutes and then twice in the Wash B solution (0.06X SSC and distillated water), centrifuged and scanned.

Data acquisition, processing and comparative phylogenomics

The scanning was carried out in an Affymatrix 418 Array Scanner (MWG Biotech) and signal data were obtained through ImaGene 5.2 (BioDiscovery). All generated data were further analysed for whole-genome comparisons of

tests strains against the L. monocytogenes EGD-e strain by GeneSpring GX 7.3.1. (Agilent Technologies UK Limited).

The signal data transformation set measurements values below 0.01 were set to 0.01. The experiment was normalized to 50th percentile of all measurements in that sample which was calculated using all genes not marked absent. For each gene represented by a signal spot the light intensity was measured dividing by control channel value in each sample. If the control channel was below 0.01 then 0.01 was used instead. If the control channel and the signal channel were both below 0.01 then no data was reported.

The presence, divergence or absence of each gene of the array was determined by GACK analysis for cut-off assignment (GACK software) by transforming the normalized data from GeneSpring in log of ratio of raw and control data (Howard et al., 2006), since this software calculate an estimated probability of presence (EPP) value for each gene (Kim et al., 2002). The relationship of the strains was determined by Mr. Bayes v.3.0 software with a Bayesian method-based algorithm (Ronquist & Huelsenbeck, 2003), however before it the GACK output trinary format was transformed into binary data which means divergent or marginal genes were converted to present. The Bayesian model employ four-chain Markov chain Monte Carlo and 16-category gamma distribution with 1 million iterations to model the presence or absence rate heterogeneity per gene throughout the genome of strain as previously described by Howard et al. (2006) and Stabler et al. (2006). Treeview was the software used to view the tree generated.

4. RESULTS AND DISCUSSION

Overall microarray analysis

A broad range of 99 L. monocytogenes strains from diverse countries and sources were used in this research (Table 1). To date 47 strains from Brazil, 11 from Denmark, 09 from Austria, 06 from Ireland, 01 from United States of America and 25 unknown origins and also a L. innocua strain, totaling 100 strains. In this set was included 58 food strains, 16 equipment and

environment of industries, 16 clinical strains (12 from human cases and 2 from animal cases), 2 strains from asymptomatic animal carriers, 6 strains from environment and 05 unknown ones (Table 1). All strains were competitively hybridized with a L. monocytogenes DNA microarray based on the whole-genome sequence L. monocytogenes EGD-e (serotype 1/2a), which has 2855 target genes.

The Bayesian algorithm allowed the clustering of the L. monocytogenes strains into 2 central clades (Figure 1) which represent the two major lineages of this species, lineage I and II as previously described (Piffareti et al., 1989), proofing the huge power of this method for strain differentiation. Additionally our study provided concordant results as earlier reported in other phylogenetic studies of L. monocytogenes genome comparison (Call et al., 2003a; Zhang et al., 2003; Doumith et al, 2004b; Nightingale et al., 2005).

Furthermore the lineage II strains were sub-divided in further two main clades, the first (clade A) bearing in its huge part serotype 1/2b strains and the other clade gathering serogroup 4 (4b and 4e serotypes) strains. This scenario has been an indicative of clustering according to L. monocytogenes lineages sub-divisions, lineage II.1 (4b, 4d and 4e) and lineage II.2 (1/2b, 3b and 7) (Doumith et al., 2004b) as shown in the figure 1. Surprisingly the clade assembling lineage II.1 strains, representing serotypes 4b and 4e split the strains in additional two sub-clades, for easier understanding we called them clades B and C, respectively. Zhang and colleagues (2003) mentioned in their research that 1/2b, 3b and 4b strains were split in more two subgroups and it was congruent with serotype distribution. Meanwhile, in our study we report, based in the comparative Bayesian approach, that lineage II strains were divided in further three sub-groups which enhances the high power of strain differentiation of microarrays allied to robust algorithm.

Based in this information and by previous findings (Nightingale et al., 2005) we can infer which lineage II strains behave as sub-populations according to its serotype since the strains serotypes 1/2b, 4b and 4e were allocated almost exclusively in separated sub-clades (A, B and C). The highly degree of clonality demonstrated by lineage II strains may be indicative of a recent population bottleneck (Meinersmann et al., 2004).

Nevertheless this pattern was not observed in lineage I strains which did not demonstrate a clear serotype outline of clustering. Hence lineage I of L. monocytogenes represent a “generalist” lineage which is better adapted to survive and multiply in the environment while still maintaining the ability to cause human disease (Nighingale et al., 2004). Call et al. (2003b) in their assessments also found that 1/2a serotype strains were distributed among three clusters with one of them being a mix of both serotypes 1/2a and 1/2c. According to Bibb et al. (1990) serotype 1/2a strains have nearly twice as much genetic variation as other L. monocytogenes serotypes.

Additionally we can deduce that lineage I and II strains represent cohesive and separate subpopulations and this hypothesis is supported by the observations of Nightingale and colleagues (2005) which agreed that horizontal genes transfer of core genes predominantly occur between strains that belong to the same lineage. Recombination should be rare event between strains belonging to different L. monocytogenes genetic lineages (Salcedo et al., 2003), indicating that L. monocytogenes lineages represents species or subspecies-like entities, with distinct ecological preferences and then a deep separation involving lineage I and II strains appears to act as a barrier to the exchange of genetic information between these two lineages (Nightingale et al., 2005).

This hypothesis can be supported making an analogy with the findings of Doumith et al. (2004b). Which comparing genomes of a lineage I (serotype 1/2a) and lineage II (serotype 4b) strains of L. monocytogenes found that 8% of lineage II strain was missing in lineage I strain even though both being of the same species. Considering and comparing the genetic diversity between two different species of Listeria, L. monocytogenes EGD-e and L. innocua, which are about are 10.5%, we can suppose L. monocytogenes lineages could be a sub-species of this bacterium. As a consequence of this divergence are the evolutionary histories and population structure of these L. monocytogenes lineages. While lineage II strains appear to be highly clonal with limited horizontal gene transfer, lineage I strains demonstrate greater genetic diversity and evidence of a larger number of horizontal gene transfer events than lineage II strains (Nightingale et al., 2005).

Aiming to know the chief divergence in the core genes of lineage I and II strains we generated a Venn diagram. This diagram was created through Genespring software analyses using only genes considered present (EPP of 100%) in the GACK analysis. Consequently it was possible to notice that lineage I and II strains share 45.5% (comprising 1293 core genes) of all 2855 genes present in the genome of sequenced L. monocytogenes EGD-e strain. The lineage I strains have 217 unique core genes in its genome while lineage II strains got 199 unique core genes.

The Genespringclustering analyses allowed the identification of specific regions present in L. monocytogenes lineages I and II strains. Thus it was found 18 specific genes for lineage I strains (Table 2 and Figure 2) which comprising five regions of L. monocytogenes genome, to date: lmo0734-lmo0739, lmo1060-lmo1063, lmo1968-lmo1971, lmo1973-lmo1974 and lmo2786-lmo2787. The functions of these genes are related to carbohydrate metabolism, two component regulatory system, ABC transporter complex and bvrB and bvrC genes. Some of these regions are considered as lineage-specific marker genes (Doumith et al., 2004b; Zhang et al., 2003) and were diagnosed like specific as well as in the lineage I strains in this study. Nelson et al. (2004) comparing whole-genome of serotype 4b (lineage II) and 1/2a strains (lineage I) found 83 genes restricted to the serotype 1/2a strains and 51 genes restricted to serotype 4b strains.

Noteworthy the genes brvB and brvC are part of a system that couples β-glucoside transport to catabolite repression of the PrfA-dependent virulence genes (Brehm et al., 1999). These genes are associated to down regulation of virulence genes in the presence of β-glucosides, which are found chiefly in plant tissues and seems to be an important feature in L. monocytogenes (Zhang et al., 2003) and according to Call et al. (2003b) this region is missing in lineage II strains, such information is in agreement to the data found in our study.

The lineage-specific marker genes for L. monocytogenes lineage II strains were only the regions lmo0466-lmo0467, lmo0469-lmo0471, lmo0151 and lmo1118-lmo1119. These regions code for hypothetical proteins and the only gene that has known function is lmo1119 which is a methylase. Maybe the complexity of identify specific markers for lineage II strains can be a failure of

this work. It may possibly be explained because the array employed in this study is based on the genome of L. monocytogenes EGD-e serotype 1/2a; turning the identification of lineage II strains specific genes complicated.

There are two simple explanations for the origin of the conserved lineage-specific and serotype-specific divergences in genome content of L. monocytogenes. First, the genes within the region of divergence (RD) may have been present in the most recent common ancestor of the two L. monocytogenes lineages and subsequently lost during divergence of lineage II. Alternatively the region could be acquired by lineage I strains, it is also possible that some can be acquired and some lost. These findings indicate that divergences between lineages I and II strains genome occurred through multiple gene acquisition and deletions events (Zhang et al., 2003). Also Nelson et al. (2004) suggested that the gene loss from a lineage ancestral to L. monocytogenes and L. innocua preceded the genomic diversification of L. monocytogenes into genomic lineages I and II. Consequently, such gene loss may have contributed to genomic improvements of L. monocytogenes, perhaps conferring high fitness to the pathogen.

Apart from the important divergence between the two main L. monocytogenes lineages, analyses identified remarkable genomic conservation within these major lineages and subgroups but variations between the different subgroups. These trails seem to mirror the evolution within the genus Listeria (Doumith et al., 2004b).

Comparative diversity among geographical origins, sources and serotypes of L. monocytogenes strains

In the clade A bearing in its biggest part serotype 1/2b strains were present strains isolated from food, equipment and environment of food industries; the only exception was the strain 1727 which is clinical strain isolated of an outbreak. It can be noted that almost every one of strains were originated from Brazil, maybe indicating epidemiologically linked strains as they were grouped as adjacent neighbours (Call et al., 2003b) in the phylogenetic tree (Figure 1).

However in the clade B gathering in its vast totality serotype 4b strains was present food and clinical strains from Brazil, Ireland and unknown origins. It can be evidence demonstrating that 4b strains besides its apparent virulence also kept features of adaptation in the environment. Food, industry equipment and asymptomatic animal strains were present into the clade C (majority serotype 4e strains), being almost exclusively grouped by strains from food origins.

Interestingly every persistent strain clustered into clade F was isolated from food and food-processing plants (industry environment). Highlighting the environment adaptation characteristic rather than the ability of cause disease of lineage I L. monocytogenes strains. Surprisingly this is the only clade where none Brazilian strains were present. However there was no evidence that geographical origin of strains was a factor determinant by which could contribute for this clustering since the persistent strains were isolated from European countries as Denmark, Austria and Ireland.

In this study, some analysed strains were studied previously by Jensen et al. (2008); where lineage I strains such as N53-1 and La111 were the less efficient causing mortality in fruit flies; 7418 and Scott A being the most efficient strains developing mortality. It is worth note that N53-1 and La111 are considered persistent strains by these authors and were also clustered in our persistent clade (clade F), while 7418 (1/2b) and Scott A (4b) both from the main clade of lineage II strains, related to disease outcome. Consequently, with these findings we may infer the real ability of strains from lineage II in causing disease, while the persistent strains are less capable to outcome disease but environmentally adapted.

Among the L. monocytogenes strains we also analysed a L. innocua strain and observing the phylogenetic tree is possible to note that L. innocua is clustered among serogroup 4 strains (clade C). This finding is in line with previous report (Zhang et al., 2003) that serotype 4b and L. innocua genome sequence are similar in some genomic regions. According to Doumith et al. (2004b) L. innocua evolved by successive gene loss from an ancestor of L. monocytogenes serogroup 4. The serogroup 4 of L. monocytogenes is closer to serogroup 6 of L. innocua than to L. monocytogenes serogroup 1/2 strains (Fiedler, 1988). There are two genes of L. innocua that are associated with

teichoic biosynthesis which were uniquely shared with L. monocytogenes strains of serogroup 4 and L. welshimeri (Doumith et al., 2004b).

Virulence genes

Virulence traits are usually considered the highest point in a pathogen. Consequently when is studied a bacterium as L. monocytogenes, considering its huge genetic diversity it is easy to believe that virulence genes should be quite variable as well. Nevertheless in our study from the 14 listed virulence genes (inlA, inlB, inlC, inlI, inlG, inlH, inlE, inlJ (precursor),uhpT, prfA, plcA, plcB, hly and actA) 85.7% were present in every studied L. monocytogenes strain (Figure 3). The exceptions were inlE and inlG genes, this last was absent mainly in lineage II strains. Interestingly there was no correlation with origin of isolation, serotype and/or geographical distribution within the presence of virulence genes. Despite of the conserved presence of virulence genes in L. monocytogenes strains, it is possible to note that gene expression regulation is the intriguing factor that trigger off the virulence profile and then the divergent profile of pathogenicity of L. monocytogenes strains.

Our results are in line with Doumith and colleagues (2004b) findings that assessing the genome content of 93 L. monocytogenes strains through microarray hybridizations found the virulence cluster (prfA, plcA, hly, mpl, actA and plcB), also inlA, inlB, uhpT and bsh genes present in all studied strains.

The internalin family proteins are surface proteins which are involved in the virulence process. In our study the internalin A was present in all strains independent from origin however only the presence is not enough to predict that strain is virulent. Based on current findings even whether persistent strains bear internalin A (inl A) gene, it does not mean they are virulent. Because single point mutations found in internalin genes from persistent strains could result in incorrect protein folding leading to a lower affinity to the E-cadherin (Jensen et al., 2008). The internalin G (inlG) was absent in the vast majority of lineage II strains, data that corroborate the findings of Doumith et al. (2004b) which reported that this gene in fact is specifically absent in lineage II strains.

According to Nelson et al. (2004) most of the Listeria virulence genes are conserved either in virulent or in less virulent Listeria strains and the presence

of these genes alone is not enough to explain the differences in virulence of any particular strain. Virulence is not a constant property and it has been speculated that it can be modulated, for instance, by components or conditions in processing of foods (reviewed by Jensen et al., 2008). For instance, in a recent research was reported that persistent RAPD type 9 strains had a lower virulence potential when compared to the clinical strains, with regards to the invasion of Caco-2 cells, killing fruits flies and nematodes worms, as well as faecal shedding and infection of guinea pig tissues (Jensen et al., 2008). Indicating which persistent strains although bearing virulence genes should keep their apparatus of virulence silent probably due some gene regulation.

Firstly our intriguing question regarding this study was whether strains isolated from different geographic regions and different sources would have similar gene content. Thus overall comparative phylogenomics of L. monocytogenes strains from Europe and Brazil have identified the two main lineages of this pathogen. Confirming that these lineages represent strains with similar genetic content independent from geographic area. Remarkably we identified a set of genes possible involved in the adaptation of strains in the environment of food industries however no significant divergences were noted among virulence genes.

5. REFERENCES

Aarts, H. J., Hakemulder, L. E., AVan Hoef., M. Genomic typing of Listeria monocytogenes strains by automated laser fluorescence analysis of amplified fragment length polymorphism fingerprint patterns. 1999. International Journal

of Food Microbiology. 49:95–102.

Bibb, W. F., Schwartz, B., Gellin, B. G., Plikaytis, B. D., Weaver, R. E. Analysis of Listeria monocytogenes by multilocus enzyme electrophoresis and application of the method to epidemiologic investigations. 1989. International

Journal of Food Microbiology. 8:233–239.

Bibb, W. F., Gellin, B. G., Weaver, R., Schwartz, B., Plikaytis, B. D., Reeves, M. W.,Pinner, R. W., Broome, C. V. Analysis of clinical and food-borne isolates of Listeria monocytogenes in the United States by multilocus enzyme electrophoresis and application of the method to epidemiologic investigations. 1990. Applied and Environmental Microbiology. 56:2133-2141

Brehm, K., Ripio, M. T., Kreft, J., Vasquez-Boland, J.-A. The bvr locus of Listeria monocytogenes mediates virulence gene expression by β-glucosides. 1999. Infection and Immunity. 181: 5024-5032.

Brosch, R., Chen, J., Luchansky, J. B. Pulsed-Field Fingerprinting of listeriae: identication of genomic divisions for Listeria monocytogenes and their correlation with serovar. 1994. Applied and Environmental Microbiology. 60: 2584-2592.

Call, D. R., Borucki, M. K., Loge, F. J. Detection of bacterial pathogens in environmental samples using DNA microarrays. 2003a. Journal of

Microbiological Methods. 53:235-243.

Call, D. R., Borucki, M. K., Besser, T. E. Mixed-genome microarrays reveal multiple serotype and lineage-specific differences among strains of Listeria monocytogenes. 2003b. Journal of Clinical Microbiology. 41:632-639.

Champion, O. L., Gaunt, M. W., Gundogdu, O., Elmi, A., Witney, A. A., Hinds, J., Dorrel, N., Wren, B. W. Comparative phylogenomics of the food-borne pathogen Campylobacter jejuni reveals genetic markers predictive of infection source. 2005. Proceedings of the National Academy of Sciences. 102:16043-16048.

Dorrell, N., Mangan, J. A., Laing, K. G., Hinds, J., Linton, D., Al-Ghusein, H., Barrell, B. G., Parkhill, J., Stoker, N. G., Karlyshev, A. V., Butcher, P. D., Wren, B. W. Whole genome comparison of Campylobacter jejuni human isolates using a low-cost microarray reveals extensive genetic diversity. 2001.

Genome Research. 11:1706–1715.

Dorrell, N., Hinchliffe, S. J., Wren, B. W. Comparative phylogenomics of the pathogenic bacteria by microarray analysis. 2005. Current Opinion in

Microbiology. 8:620-626.

Doumith, M., Buchrieser, C., Glaser, P., Jacquet, C. and Martin, P. Differentiation of the major Listeria monocytogenes serovars by multiplex PCR. 2004a. Journal of Clinical Microbiology. 42:3819-3822.

Doumith, M., Cazalet, C., Simoes, N., Frangeul, L., Jacquet, C., Kunst, F., Martin, P., Cossart, P., Glaser, P., Buchrieser, C. New aspects regarding evolution and virulence of Listeria monocytogenes revealed by comparative genomics and DNA arrays. 2004b. Infection and Immunity. 72:1072-1083. Dumas, E., Meunier, B., Berdagué, J.-L., Chambon, C. Comparative analysis of extracellular and intracellular proteomes of Listeria monocytogenes strains reveals a correlation between protein expression and serovar. 2008. Applied

and Environmental Microbiology. 74:7399-7409.

European Food Safety Authority (EFSA). 2007. The community summary

resistance and foodborne outbreaks in the European Union in 2006. Euro Food Safety Authority. J. 130:1-310.

Fiedler, F. Biochemistry of the cell surface of Listeria strains: a locating general view. 1988. Infection. 16:92-97.

Gillespie, I. A., McLauchlin, J., Grant, K. A., Little, C. L., Mithani, V., Penman, C., Lane, C.; Regan, M. 2006. Emerging Infectious Diseases. 12:1361-1366. Glaser, P. Frangeul, L., Buchrieser, C., Rusniok, C., Amend, A., Baquero, F., Berche, P., Bloecker, H., Brandt, P., Chakraborty, T., Charbit, A., Chetouani, F., Couvé, E., de Daruvar, A., Dehoux, P., Domann, E., Domínguez-Bernal, G., Duchaud, E., Durant, L., Dussurget, O., Entian, K.-D., Fsihi, H., Garcia-Del Portillo, F., Garrido, P., Gautier, L., Goebel, W., Gómez-López, N., Hain, T., Hauf, J., Jackson, D., Jones, L.-M., Kaerst, U., Kreft, J., Kuhn, M., Kunst, F., Kurapkat, G., Madueño, E., Maitournam, A., Mata Vicente, J., Ng, E., Nedjari, H., Nordsiek, G., Novella, S., de Pablos, B., Pérez-Diaz, J.-C., Purcell, R., Remmel, B., Rose, M., Schlueter, T., Simoes, N., Tierrez, A., Vázquez-Boland, J.-A., Voss, H., Wehland, J., and Cossart, P. Comparative Genomics of Listeria Species. 2001. Science. 294:849 – 852.

Goulet, V., Hedberg, C., Le Monnier, A., Valk, H. Increasing incidence of listeriosis in France and other European countries. 2008. Emerging Infectious

Diseases. 14: 734-740.

Hinds, J., Laing, K. G., Mangan, J. A., Butcher, P. D. Microarrays for microbes: the BµG@S approach. 2002. Comparative and Functional Genomics. 3:333-337.

Hofer, E., Ribeiro, R., Feitosa, D. P. Species and serovars of the genus Listeria isolated from different sources in Brazil from 1971 to 1997. 2000. Memórias do

Instituto Oswaldo Cruz. 95:615-620.

Howard, S. L., Gaunt, M. W., Hinds, J. Witney, A. A., Stabler, R., Wren, B. W. Application of comparative phylogenomics to study the evolution of Yersinia enterocolitica and to identify genetic differences relating to pathogenicity. 2006.

Journal of Bacteriology. 188:3645-3653.

Jensen, A., Thomsen, L. E., Jorgensen, R. L., Larsen, M. H., Roldgaard, B. B., Christensen, B. B., Vogel, B. F., Gram, L., Ingmer, H. Processing plant persistent strains of Listeria monocytogenes appear to have a lower virulence potential than clinical strains in selected virulence models. 2008. International

Journal of Food Microbiology. 123:254-261.

Kathariou, S. Listeria monocytogenes virulence and pathogenicity, a food safety perspective. 2002. Journal of Food Protection., 5: 1811-1829.

Kim, C. C., Joyce, E. A., Chan, K., Falkow, S. Improved analytical methods for microarray-based genome-composition analysis. 2002. Genome Biology. 3:RESEARCH0065.

Lima, A. S., Laer, A. E., Trindade, P. S., Silva, W. P. Disseminação de Listeria monocytogenes no processamento de lingüiça mista frescal avaliada por sorologia e RAPD. 2005. Alimentos e Nutrição. 16:245-251.

Mead, P. S., L. Slutsker, V. Dietz. L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Griffin; Tauxe, R. V. 1999. Food-related illness and death in the United States.

Emerging Infectious Diseases. 5:607-625.

Meinersmann, R. J., Phillips, R. W., Wiedmann, M., Berrang, M. Multilocus sequence typing of Listeria monocytogenes by use of hypervariable genes reveals clonal and recombination histories of three lineages. 2004. Applied and

Environment Microbiology. 70:2193-2203.

Miya, S., Takahashi, H., Ishikawa, T., Fujii, T., Kimura. Brisk of Listeria monocytogenes contamination of raw ready-to-eat seafood products available at retail outlets in Japan. 2010. Applied and Environmental Microbiology. 76:3383-3386.

Nalério, E. N., Araújo, M. R., Mendonça, K. S., Bassani, M. T., Silva, W. P. Listeria monocytogenes: monitoramento desse perigo biológico na cadeia produtiva de frangos do sul do Rio Grande do Sul. 2009. Ciência e Tecnologia

de Alimentos. 29.

Nelson, K. E., Fouts, D. E., Mongodin, E. F., Ravel, J., DeBoy, R. T., Kolonay, J. F., Rasko, D. A., Angiuoli, S. V., Gill, S.R., Paulsen, I. T., Peterson, J., White, O., Nelson, W. C., Nierman, W., Beanan, M. J., Brinkac, L. M., Daugherty, S. C., Dodson, R. J., Durkin, A. S., Madupu, R., Haft, D. H., Selengut, J., Aken, S. V., Khouri, H., Fedorova, N., Forberger, H., Tran, B., Kathariou, S., Wonderling, L. D., Uhlich, G. A., Bayles, D. O., Luchansky, J. B., Fraser, C. M. Whole genome comparisons of serotype 4b and 1/2a

strains of the food-borne pathogen Listeria monocytogenes reveal new insights into the core genome components of this species. 2004. Nucleic Acids

Research. 32:2386-2395.

Nightingale, K. K., Schukken, Y. H. Nightingale C. R., Fortes, E. D., Ho, A. J. Her, Z. Grohn, Y. T., McDonough, P. L. Wiedmann. M. 2004. Ecology and transmission of Listeria monocytogenes in ruminants and the farm environment.

Applied and Environment Microbiology. 70:4458–4467

Nightingale, K. K., Windham, K., Wiedmann, M. Evolution and molecular phylogeny of Listeria monocytogenes isolated from human and animal listeriosis cases and food. 2005. Journal of Bacteriology. 187: 5537-5551.

Piffaretti, J. C., Kressebuch, H., Aeschbacher, M., Bille, J., Bannerman, J. M., Selander, R. K., Rocourt, J. 1989. Genetic characterization of clones of the bacterium Listeria monocytogenes causing epidemic disease. Proceedings of

Ronquist, F.; Huelsenbeck, J. P. MrBayes3: Bayesian phylogenetic inference under mixed models. 2003. Bioinformatics. 19:1572-1574.

Rozen, S. & Skaletsky, H. 2000. Methods in Molecular Biology.132, 365-386. Salcedo, C., Arreaza L., Alcala, B., de la Fuente L., Vazquez J. A.. 2003. Development of a multilocus sequence typing method for analysis of Listeria monocytogenes clones. Journal of Clinical Microbiology. 41:757–762.

Schlech, W. F. 1998. Foodborne listeriosis. Clinical Infectious Disease. 31:770-775.

Shuchat, A., Swaminathan, B. and Broome, C. 1991. Epidemiology of human listeriosis. Clinical Microbiology Reviews. 4:169-183.

Stabler, R. A., Gerding, D. N., Songer, J. G., Drudy, D., Brazier, J.S., Trinh, H. T., Witney, A. A., Hinds, J., Wren, B. W. Comparative phylogenomics of Clostridium difficile reveals clade specificity and microevolution of hypervirulent strains. Journal of Bacteriology. 2006. 188:7297-7305.

Hellström, S., Laukkanen, R., Siekkinen, K. M., Ranta, J., Maijala, R., Korkeala, H. Listeria monocytogenes contamination in pork can originate from farms.

Food Protection. 2010. 73:641-648.

Wiedmann, M. 2002. Molecular subtyping methods for Listeria monocytogenes.

Journal of AOAC International. 85:524-531.

Zhang, C., Zhang, M., Ju, J. Nietfeltdt, J., Wise, J., Terry, P. M., Olson, M., Kachmann, S. D., Wiedmann, M., Samadpour, M., Benson, A. K. Genome diversification in phylogenetic lineages I and II of Listeria monocytogenes: identification of segments unique to lineage II populations. 2003. Journal of

Table 1 – Identification and information about strains

Strain Origin

Year of

isolation Group

Serotype/

Serogroup Lineage City -Country

19 Cheese 2007 Food - - Cork - Ireland

30 Dry faeces – cow - Animal asymptomatic 4b II-1 Cork - Ireland

92 Cheese - Food - - Cork - Ireland

411 Unknown - Unknown - - Cork - Ireland

535 Vacherin Cheese 1996 Food - - Unknown

758 Producer B – smear 1997 Environment – Persistent - - Vienna - Austria 1454 Clinical – animal (cow – mastitis) 1997 Animal 1/2a I-1 Vienna - Austria 1458 Producer A – smear 1997 Environment – non-persistent - - Vienna - Austria

1727 Outbreak 1998 Human - - Atlanta, CDC - USA

2315 Producer B – Cheese 1999 Food- Persistent - - Vienna - Austria 2540 Producer A - Cheese rasp 1999 Food – Persistent - - Vienna - Austria 2566 Producer B – smear 1999 Environment - non-persistent - - Vienna - Austria

4239 Human case ( 52 year-old male) 1998 Human 1/2a I-1 Unknown

4359 Producer A - water 2004 Food – Persistent - - Vienna - Austria 4423 Producer B – smear 2004 Environment - Persistent - - Vienna - Austria 4446 Clinical – Human case ( 63 year-old female) 1998 Human 4b II-1 Unknown 4542 Clinical – Human case ( 84 year-old male) 1998 Human 4b II-1 Unknown 4810 Clinical – Human case ( 84 year-old male) 1998 Human 4 II Unknown

4898 Producer A - water 2007 Food – Persistent - - Vienna - Austria

4905 Human case ( 70 year-old female) 1998 Human 1/2 - Unknown

6179 Cheese 2000 Food – Persistent 1/2a I-1 Cork - Ireland

6895 Ham 1998 Food 1/2 II Unknown

7291 Pasta with chicken 1998 Food 4b II-1 Unknown

7418 Spreadable sausage 1998 Food – Sporadic 1/2b II-2 Unknown

2V903b Smokehouse V - cold-smoked salmon - final product - plant 1 1999 Food 4 I Denmark 3R23-1 Smokehouse 1 – steel 2003 Industry Environment - II Denmark Ag28 Seawater fish farm 1 - stone with algae 2004 Environment - Sporadic - - Unknown Ap143 Slaughterhouse 2 - mud from bleeding area 2004 Industry Environment - - Unknown Br21 Fresh water fish farm 2 - water from detesting tank 2004 Environment - Persistent 1/2a I-1 Unknown Br22 Fresh water fish farm 2 - sediment from detesting tank 2004 Environment - - Unknown BRA 1 Rubber plucker fingers of defeathering machine 2005 Industry Environment 4b II-1 Pelotas - Brazil

Table1 - Continuation

BRA 102 Chilled chicken 2006 Food – Retail 1/2b II-2 Pelotas - Brazil

BRA 104 Chilled chicken 2006 Food – Retail 1/2a I-1 Pelotas - Brazil

BRA 107 Chilled chicken 2006 Food – Retail 1/2b II-2 Pelotas - Brazil

BRA 108 Chilled chicken 2006 Food – Retail 1/2a I-1 Pelotas - Brazil

BRA 10s Pork sausage - Food 1b - Pelotas - Brazil BRA 111 Chilled chicken 2006 Food – Retail 1/2b II-2 Pelotas - Brazil BRA 11o Sheep carcass - Food 4b II-1 Pelotas - Brazil BRA 12o Sheep carcass - Food 1/2c I-2 Pelotas - Brazil BRA 13s Pork sausage - Food 4b II-1 Pelotas - Brazil BRA 18o Sheep carcass - Food 1/2b II-2 Pelotas - Brazil BRA 1s Pork sausage - Food 4b II-1 Pelotas - Brazil BRA 20 Evisceration floor 2006 Industry Environment 1/2b II-2 Pelotas - Brazil BRA 21o Sheep carcass - Food 4b II-1 Pelotas - Brazil BRA 22 Washing water prechiller 2006 Industry Environment 1/2b II-2 Pelotas - Brazil BRA 22o Sheep carcass - Food 1/2c I-2 Pelotas - Brazil BRA 22q Cottage cheese - Food – retail 1/2a I-1 Pelotas - Brazil BRA 23q Cottage cheese - Food – retail 4b II-1 Pelotas - Brazil BRA 24q Cottage cheese - Food – retail 1/2b II-2 Pelotas - Brazil BRA 25s Handling (pork sausage) - - 1c - Pelotas - Brazil BRA 27 Washing water chiller 2006 Industry Environment 1/2b II-2 Pelotas - Brazil BRA 2o Sheep carcass - Food 4b II-1 Pelotas - Brazil BRA 32 Conveyor cut belt 2006 Industry Environment 1/2b II-2 Pelotas - Brazil BRA 42 Chicken Final product 2006 Food - Industry Environment 1/2b II-2 Pelotas - Brazil BRA 45 Washing water chicken from evisceration 2006 Industry Environment 1/2b II-2 Pelotas - Brazil BRA 46 Evisceration floor 2006 Industry Environment 1/2b II-2 Pelotas - Brazil BRA 53 Conveyor cut belt 2006 Industry Environment 1/2b II-2 Pelotas - Brazil

BRA 58 Chilled chicken 2006 Food - retail 4e II-1 Pelotas - Brazil

BRA 63 Chilled chicken 2006 Food -retail 4e II-1 Pelotas - Brazil

BRA 6o Sheep carcass - Food 3c I-2 Pelotas - Brazil

BRA 71 Chilled chicken 2006 Food - Retail 4e II-1 Pelotas - Brazil

BRA 74 Chilled chicken 2006 Food - Retail 4e II-1 Pelotas - Brazil

BRA 76 Chicken Final product 2006 Food – Industry Environment 4e II-1 Pelotas - Brazil

BRA 77 Chilled chicken 2006 Food – Retail 4e II-1 Pelotas - Brazil

BRA 82 Chilled chicken 2006 Food – Retail 4e II-1 Pelotas - Brazil

BRA 86 Chicken cloacae swab (faeces) 2006 Animal asymptomatic 4e II-1 Pelotas - Brazil

Table 1 - Continuation

BRA 8q Cottage cheese - Food – Retail 1/2c I-2 Pelotas - Brazil BRA 8s Pork sausage - Food – Retail 1c - Pelotas - Brazil

BRA 91 Chilled chicken 2006 Food – Retail 1/2a I-1 Pelotas - Brazil

BRA 94 Chilled chicken 2006 Food – Retail 1/2b II-2 Pelotas - Brazil

BRA 98 Chilled chicken 2006 Food – Retail 1/2c I-2 Pelotas - Brazil

BRA 99 Chilled chicken 2006 Food – Retail - - Pelotas - Brazil

BRA 9o Sheep carcass - Food 1/2a I-1 Pelotas - Brazil BRA

St07 Clinical – human - Human 1/2b II-2 Brazil

BRA

St08 Clinical – human - Human 1/2a I-1 Brazil

BRA

St247 Clinical – human - Human 4b II-1 Brazil

EDG Animal case – rabbit 1926 Animal - Standard strain 1/2a I-1 Unknown H025 Fresh water fish farm 1 - surface of rainbow trout 2004 Environment - - Unknown

L. innocua - - - 6a - Unknown

La111 Smokehouse G - cold-smoked salmon 1996 Food – Industry environment 1/2a I-1 Denmark La22 Smokehouse V - cold-smoked salmon - final product 1995 Food - Industry environment 1/2a I-1 Denmark

11994 Unknown - Unknown - - Unknown

Lm 7973 Unknown - Unknown - - Unknown

Lo28 Clinical - Pregnant woman - Human Faeces 1/2c I-2 Unknown M103-1 Slaughterhouse A - rubber conveyor belt 2003 Industry Environment 1/2a I-1 Denmark N22-2 Smokehouse 2 - floor in slicing area 2002 Sporadic - food industry environment - - Unknown N53-1 Smokehouse 2 - scraper mat 2002 Industry Environment 1/2a I-1 Denmark No37-1 Smokehouse 3 - waste container (pet food) 2001 Industry Environment - II Denmark No40-1 Smokehouse 3 - floor swap 2003 Industry Environment 1/2a I-1 Denmark R416a Smokehouse 1 - slicing machine - processing plant 2 1996 Industry Environment - - Denmark R479a Smokehouse 1 - cold-smoked salmon - final product - processing plant 2 1996 Food – Persistent 1/2a I-1 Denmark S2583 Lettuce processing equipment 1998 Industry Environment 1/2 II Unknown

Scott A Human - Human – Standard strain 4b II-1 Unknown

SVS2719 Animal case – goat 1998 Animal 1/2 II Unknown

SVS2827 Ensilage 1998 Food 1/2 II Unknown

V518a

Smokehouse V - cold-smoked salmon- final product slicing mach. 2 - proc

plant 1 1998 Food - Industry Environment 4b II-1 Denmark

Vo14 Fresh water fish farm 3 - grass and plants around the race-way-system 2004 Environment - - Unknown Vo26 Fresh water fish farm 3 - sediment from incoming stream 2004 Environmentl - - Unknown 29 Unknown - Unknown - - Cork - Ireland

Figure 1 – Phylogenetic tree showing the genetic relationship among L. monocytogenes strains. A – clade of majority 1/2b strains; B – clade of majority 4b strains; C – clade of majority 4e strains; D, E and F – clade of majority 1/2a.

Table 2 - Specific-gene markers of L. monocytogenes lineage I strains Gene

ID

Gene function

Lmo0734 Similar to transcriptional regulator (LacI family)

Lmo0735 Similar to ribulose 5- phosphate 3-epimerase

Lmo0736 Similar to ribose 5-phosphate isomerise

Lmo0737 Conserved hypothetical protein

Lmo0738 Similar to phosphotransferase system (PTS) beta-glucoside-specific enzyme II ABC component

Lmo0739 Similar to 6-phospho-beta-glucosidase

Lmo1060 Similar to transcription response regulator

Lmo1061 Similar to two-component sensor histidine kinase

Lmo1062 Similar to ABC transporters (permease protein)

Lmo1063 Similar to ABC transporter (ATP binding protein)

Lmo1968 Similar to creatinine amidohydrolases

Lmo1969 similar to 2-keto-3-deoxygluconate-6-phosfate aldolase

Lmo1970 similar to putative to phosphotriesterase related protein

Lmo1971 Similar to pentitol PTS system enzyme II C component

Lmo1973 Similar to PTS system enzyme II A component

Lmo1974 Similar to transcriptions regulators (GntR family)

Lmo2786 bvrC

Figure 2 – Genespring clustering of lineage I and II strains. Yellow bars represent present genes and blue bars represent absent genes in the strains.

Figure 3 – GeneSpring clustering of virulence genes of L. monocytogenes, yellow bars indicating present genes and blue bars absent genes.

TÍTULO 2: Comparative phylogenomics of Listeria monocytogenes reveals specific genes related to adaptation profile